Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.     

Tuning dynamic DNA- and peptide-driven self-assembly in DNA–peptide conjugates

DNA–peptide conjugates offer an opportunity to marry the benefits of both biomolecular classes, combining the high level of programmability found with DNA, with the chemical diversity of peptides. These hybrid systems offer potential in fields such as therapeutics, nanotechnology, and robotics. Using the first DNA–β-turn peptide conjugate, we present three studies investigating the self-assembly of DNA–peptide conjugates over a period of 28 days. Time-course studies, such as these have not been previously conducted for DNA–peptide conjugates, although they are common in pure peptide assembly, for example in amyloid research. By using aging studies to assess the structures produced, we gain insights into the dynamic nature of these systems. The first study explores the influence varying amounts of DNA–peptide conjugates have on the self-assembly of our parent peptide. Study 2 explores how DNA and peptide can work together to change the structures observed during aging. Study 3 investigates the presence of orthogonality within our system by switching the DNA and peptide control on and off independently. These results show that two orthogonal self-assemblies can be combined and operated independently or in tandem within a single macromolecule, with both spatial and temporal effects upon the resultant nanostructures.

DNA and peptide nanotechnologies can be interfaced to create hierarchical and emergent superstructures, which evolve with time.     

Design,Synthetic Strategies,and Therapeutic Applications of Heterofunctional Glycodendrimers

Glycodendrimers have attracted considerable interest in the field of dendrimer sciences owing to their plethora of implications in biomedical applications. This is primarily due to the fact that cell surfaces expose a wide range of highly diversified glycan architectures varying by the nature of the sugars, their number, and their natural multiantennary structures. This particular situation has led to cancer cell metastasis, pathogen recognition and adhesion, and immune cell communications that are implicated in vaccine development. The diverse nature and complexity of multivalent carbohydrate–protein interactions have been the impetus toward the syntheses of glycodendrimers. Since their inception in 1993, chemical strategies toward glycodendrimers have constantly evolved into highly sophisticated methodologies. This review constitutes the first part of a series of papers dedicated to the design, synthesis, and biological applications of heterofunctional glycodendrimers. Herein, we highlight the most common synthetic approaches toward these complex molecular architectures and present modern applications in nanomolecular therapeutics and synthetic vaccines.     

Molecular heterogeneity analysis of poly(amidoamine) dendrimer-based mono- and multifunctional nanodevices by capillary electrophoresis

Poly(amidoamine) (PAMAM) dendrimer-based nanodevices are of recent interest in targeted cancer therapy. Characterization of mono- and multifunctional PAMAM-based nanodevices remains a great challenge because of their molecular complexity. In this work, various mono- and multifunctional nanodevices based on PAMAM G5 (generation 5) dendrimer were characterized by UV-Vis spectrometry, (1)H NMR, size exclusion chromatography (SEC), and capillary electrophoresis (CE). CE was extensively utilized to measure the molecular heterogeneity of these PAMAM-based nanodevices. G5-FA (FA denotes folic acid) conjugates (synthesized from amine-terminated G5.NH(2) dendrimer, approach 1) with acetamide and amine termini exhibit bimodal or multi-modal distributions. In contrast, G5-FA and bifunctional G5-FA-MTX (MTX denotes methotrexate) conjugates with hydroxyl termini display a single modal distribution. Multifunctional G5.Ac(n)-FI-FA, G5.Ac(n)-FA-OH-MTX, and G5.Ac(n)-FI-FA-OH-MTX (Ac denotes acetamide; FI denotes fluorescein) nanodevices (synthesized from partially acetylated G5 dendrimer, approach 2) exhibit a monodisperse distribution. It indicates that the molecular distribution of PAMAM conjugates largely depends on the homogeneity of starting materials, the synthetic approaches, and the final functionalization steps. Hydroxylation functionalization of dendrimers masks the dispersity of the final PAMAM nanodevices in both synthetic approaches. The applied CE analysis of mono- and multifunctional PAMAM-based nanodevices provides a powerful tool to evaluate the molecular heterogeneity of complex dendrimer conjugate nanodevices for targeted cancer therapeutics.     

A photoresponsive antibody–siRNA conjugate for activatable immunogene therapy of cancer

Tumor-targeted delivery of small-interfering RNAs (siRNAs) for cancer therapy still remains a challenging task. While antibody–siRNA conjugates (ARCs) provide an alternative way to address this challenge, the uncontrollable siRNA release potentially leads to undesirable off-tumor side effects, limiting their in vivo therapeutic efficacy. Here, we report a photoresponsive ARC (PARC) for tumor-specific and photoinducible siRNA delivery as well as photoactivable immunogene therapy. PARC is composed of an anti-programmed death-ligand 1 antibody (αPD-L1) conjugated with a siRNA against intracellular PD-L1 mRNA through a photocleavable linker. After targeting cancer cells through the interaction between αPD-L1 and membrane PD-L1, PARC is internalized and it liberates siPD-L1 upon light irradiation to break the photocleavable linker. The released siPD-L1 then escapes from the lysosome into the cytoplasm to degrade intracellular PD-L1 mRNA, which combines the blockade of membrane PD-L1 by αPD-L1 to boost immune cell activity. Owing to these features, PARC causes effective cancer suppression both in vitro and in vivo. This study thus provides a useful conditional delivery platform for siRNAs and a novel means for activatable cancer immunogene therapy.

A photoresponsive antibody–siRNA conjugate (PARC) enables tumor-targeted siRNA delivery and photoactivatable gene silencing for cancer immunotherapy.     

A water-soluble aromatic dendrimer as a model basis for dual-action drugs

The affinities of two anionic pyrenyl probes for pyridinium high-molecular-mass cations of different topologies—poly(N-ethyl-4-vinylpyridinium bromide) and a water-soluble poly(pyridylphenylene) dendrimer—are studied by the method of fluorescence quenching. The hydrophilic probe carrying three sulfonate groups in a molecule more efficiently interacts with a flexible highly charged linear polycation throughout the studied pH range. The binding of the dendrimer with a relatively hydrophobic probe containing a single carboxyl group is improved by acidification of solutions, and it becomes dominant in weakly acidic solutions. The interaction of DNA with the dendrimer containing the hydrophobic probe has no effect on the formation of the dendriplex and leads to displacement of only a small fraction of the bound probe into solution. Our model studies demonstrate that dual-action dendrimer carriers capable of simultaneous delivery of genetic material and hydrophobic drugs to target cells can be created.     

Charge stabilization via electron exchange: excited charge separation in symmetric,central triphenylamine derived,dimethylaminophenyl–tetracyanobutadiene donor–acceptor conjugates

Photoinduced charge separation in donor–acceptor conjugates plays a pivotal role in technology breakthroughs, especially in the areas of efficient conversion of solar energy into electrical energy and fuels. Extending the lifetime of the charge separated species is a necessity for their practical utilization, and this is often achieved by following the mechanism of natural photosynthesis where the process of electron/hole migration occurs distantly separating the radical ion pairs. Here, we hypothesize and demonstrate a new mechanism to stabilize the charge separated states via the process of electron exchange among the different acceptor entities in multimodular donor–acceptor conjugates. For this, star-shaped, central triphenylamine derived, dimethylamine–tetracyanobutadiene conjugates have been newly designed and characterized. Electron exchange was witnessed upon electroreduction in conjugates having multiple numbers of electron acceptors. Using ultrafast spectroscopy, the occurrence of excited state charge separation, and the effect of electron exchange in prolonging the lifetime of charge separated states in the conjugates having multiple acceptors have been successfully demonstrated. This work constitutes the first example of stabilizing charge-separated states via the process of electron exchange.

The significance of electron exchange in stabilizing the charge-separated state is revealed in multi-modular donor–acceptor conjugates.     

Application of Dendrimers in Anticancer Diagnostics and Therapy

The application of dendrimeric constructs in medical diagnostics and therapeutics is increasing. Dendrimers have attracted attention due to their compact, spherical three-dimensional structures with surfaces that can be modified by the attachment of various drugs, hydrophilic or hydrophobic groups, or reporter molecules. In the literature, many modified dendrimer systems with various applications have been reported, including drug and gene delivery systems, biosensors, bioimaging contrast agents, tissue engineering, and therapeutic agents. Dendrimers are used for the delivery of macromolecules, miRNAs, siRNAs, and many other various biomedical applications, and they are ideal carriers for bioactive molecules. In addition, the conjugation of dendrimers with antibodies, proteins, and peptides allows for the design of vaccines with highly specific and predictable properties, and the role of dendrimers as carrier systems for vaccine antigens is increasing. In this work, we will focus on a review of the use of dendrimers in cancer diagnostics and therapy. Dendrimer-based nanosystems for drug delivery are commonly based on polyamidoamine dendrimers (PAMAM) that can be modified with drugs and contrast agents. Moreover, dendrimers can be successfully used as conjugates that deliver several substances simultaneously. The potential to develop dendrimers with multifunctional abilities has served as an impetus for the design of new molecular platforms for medical diagnostics and therapeutics.     

Design of peptide–dendrimer conjugates with tumor homing and antitumor effects

Development of drug delivery systems for cancer therapy is a crucial issue. Previously, some peptides were designed as tumor homing cell-penetrating peptides with antitumor activities. In this study, dual function dendrimers with tumor targeting activities and antitumor effects were designed using the tumor targeting CPP44 peptide for acute myelogenous leukemia (AML) and the antitumor p16^INK4a peptide. Two types of peptide–dendrimer conjugates were synthesized. One was a CPP44-linked p16^INK4a peptide-conjugated dendrimer (tandem linked dendrimer) and the other was a dendrimer conjugated with separate CPP44 and p16^INK4a peptides (parallel linked dendrimer). In addition, a peptide cathepsin B substrate was linked to the antitumor p16^INK4a peptide to release it from the carriers. These peptide–dendrimer conjugates produced more effective antitumor effects than a CPP44-linked p16^INK4a peptide. The parallel linked dendrimer showed less association with AML cells than the tandem linked dendrimer, but had greater antitumor effects. This suggested that both cellular uptake and antitumor peptide cleavage affected the antitumor activities of dual functional peptide-conjugated dendrimers.     

Curcumin Loaded Dendrimers Specifically Reduce Viability of Glioblastoma Cell Lines

Glioblastoma (GB) is a deadly and aggressive cancer of the CNS. Even with extensive resection and chemoradiotherapy, patient survival is still only 15 months. To maintain growth and proliferation, cancer cells require a high oxidative state. Curcumin, a well-known anti-inflammatory antioxidant, is a potential candidate for treatment of GB. To facilitate efficient delivery of therapeutic doses of curcumin into cells, we encapsulated the drug in surface-modified polyamidoamine (PAMAM) dendrimers. We studied the in vitro effectiveness of a traditional PAMAM dendrimer (100% amine surface, G4 NH₂), surface-modified dendrimer (10% amine and 90% hydroxyl-G4 90/10-Cys), and curcumin (Cur)-encapsulated dendrimer (G4 90/10-Cys-Cur) on three species of glioblastoma cell lines: mouse-GL261, rat-F98, and human-U87. Using an MTT assay for cell viability, we found that G4 90/10-Cys-Cur reduced viability of all three glioblastoma cell lines compared to non-cancerous control cells. Under similar conditions, unencapsulated curcumin was not effective, while the non-modified dendrimer (G4 NH₂) caused significant death of both cancerous and normal cells. By harnessing and optimizing the components of PAMAM dendrimers, we are providing a promising new route for delivering cancer therapeutics. Our results with curcumin suggest that antioxidants are good candidates for treating glioblastoma.     

A comprehensive analysis in one run – in-depth conformation studies of protein–polymer chimeras by asymmetrical flow field-flow fractionation

Polymer-based protein engineering has enabled the synthesis of a variety of protein–polymer conjugates that are widely applicable in therapeutic, diagnostic and biotechnological industries. Accurate characterizations of physical–chemical properties, in particular, molar masses, sizes, composition and their dispersities are critical parameters that determine the functionality and conformation of protein–polymer conjugates and are important for creating reproducible manufacturing processes. Most of the current characterization techniques suffer from fundamental limitations and do not provide an accurate understanding of a sample''s true nature. In this paper, we demonstrate the advantage of asymmetrical flow field-flow fractionation (AF4) coupled with multiple detectors for the characterization of a library of complex, zwitterionic and neutral protein–polymer conjugates. This method allows for determination of intrinsic physical properties of protein–polymer chimeras from a single, rapid measurement.

Precise characterization of structural parameters and their polydispersities of protein–polymer conjugates is performed with rapid analysis using asymmetrical flow field-flow fractionation coupled with multiple detectors.     

Targeting virulence: salmochelin modification tunes the antibacterial activity spectrum of β-lactams for pathogen-selective killing of Escherichia coli

New antibiotics are required to treat bacterial infections and counteract the emergence of antibiotic resistance. Pathogen-specific antibiotics have several advantages over broad-spectrum drugs, which include minimal perturbation to the commensal microbiota. We present a strategy for targeting antibiotics to bacterial pathogens that utilises the salmochelin-mediated iron uptake machinery of Gram-negative Escherichia coli. Salmochelins are C-glucosylated derivatives of the siderophore enterobactin. The biosynthesis and utilisation of salmochelins are important for virulence because these siderophores allow pathogens to acquire iron and evade the enterobactin-scavenging host-defense protein lipocalin-2. Inspired by the salmochelins, we report the design and chemoenzymatic preparation of glucosylated enterobactin–β-lactam conjugates that harbour the antibiotics ampicillin (Amp) and amoxicillin (Amx), hereafter GlcEnt–Amp/Amx. The GlcEnt scaffolds are based on mono- and diglucosylated Ent where one catechol moiety is functionalized at the C5 position for antibiotic attachment. We demonstrate that GlcEnt–Amp/Amx provide up to 1000-fold enhanced antimicrobial activity against uropathogenic E. coli relative to the parent β-lactams. Moreover, GlcEnt–Amp/Amx based on a diglucosylated Ent (DGE) platform selectively kill uropathogenic E. coli that express the salmochelin receptor IroN in the presence of non-pathogenic E. coli and other bacterial strains that include the commensal microbe Lactobacillus rhamnosus GG. Moreover, GlcEnt–Amp/Amx evade the host-defense protein lipocalin-2, and exhibit low toxicity to mammalian cells. Our work establishes that siderophore–antibiotic conjugates provide a strategy for targeting virulence, narrowing the activity spectrum of antibiotics in clinical use, and achieving selective delivery of antibacterial cargos to pathogenic bacteria on the basis of siderophore receptor expression.     

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids

Development of multiple chemical tools for deoxyribonucleic acid (DNA) labeling has facilitated wide use of their functionalized conjugates, but significant practical and methodological challenges remain to achievement of site-specific chemical modification of the biomacromolecule. As covalent labeling processes are more challenging in aqueous solution, use of nonaqueous, biomolecule-compatible solvents such as an ionic liquid consisting of a salt with organic molecule architecture, could be remarkably helpful in this connection. Herein, we demonstrate site-specific chemical modification of unprotected DNAs through a tetrazene-forming amine–azide coupling reaction using an ionic liquid. This ionic liquid-enhanced reaction process has good functional group tolerance and precise chemoselectivity, and enables incorporation of various useful functionalities such as biotin, cholesterol, and fluorophores. A site-specifically labeled oligonucleotide, or aptamer interacting with a growth factor receptor (Her2) was successfully used in the fluorescence imaging of breast cancer cell lines. The non-traditional medium-promoted labeling strategy described here provides an alternative design paradigm for future development of chemical tools for applications involving DNA functionalization.

Site-specific chemical modification of unprotected DNAs through a phosphine-mediated amine–azide coupling reaction in ionic liquid.     

Molecularly Precise Dendrimer–Drug Conjugates with Tunable Drug Release for Cancer Therapy

The structural preciseness of dendrimers makes them perfect drug delivery carriers, particularly in the form of dendrimer–drug conjugates. Current dendrimer–drug conjugates are synthesized by anchoring drug and functional moieties onto the dendrimer peripheral surface. However, functional groups exhibiting the same reactivity make it impossible to precisely control the number and the position of the functional groups and drug molecules anchored to the dendrimer surface. This structural heterogeneity causes variable pharmacokinetics, preventing such conjugates to be translational. Furthermore, the highly hydrophobic drug molecules anchored on the dendrimer periphery can interact with blood components and alter the pharmacokinetic behavior. To address these problems, we herein report molecularly precise dendrimer–drug conjugates with drug moieties buried inside the dendrimers. Surprisingly, the drug release rates of these conjugates were tailorable by the dendrimer generation, surface chemistry, and acidity.     

Structure-guided design of CPPC-paired disulfide-rich peptide libraries for ligand and drug discovery

Peptides constrained through multiple disulfides (or disulfide-rich peptides, DRPs) have been an emerging frontier for ligand and drug discovery. Such peptides have the potential to combine the binding capability of biologics with the stability and bioavailability of smaller molecules. However, DRPs with stable three-dimensional (3D) structures are usually of natural origin or engineered from natural ones. Here, we report the discovery and identification of CPPC (cysteine–proline–proline–cysteine) motif-directed DRPs with stable 3D structures (i.e., CPPC–DRPs). A range of new CPPC–DRPs were designed or selected from either random or structure–convergent peptide libraries. Thus, for the first time we revealed that the CPPC–DRPs can maintain diverse 3D structures by taking advantage of constraints from unique dimeric CPPC mini-loops, including irregular structures and regular α-helix and β-sheet folds. New CPPC–DRPs that can specifically bind the receptors (CD28) on the cell surface were also successfully discovered and identified using our DRP-discovery platform. Overall, this study provides the basis for accessing an unconventional peptide structure space previously inaccessible by natural DRPs and computational designs, inspiring the development of new peptide ligands and therapeutics.

CPPC-paired disulfide-rich peptides with stable 3D structures have been discovered through rational library design and screening, providing unconventional peptide scaffolds for the development of new peptide therapeutics.     

Inhibition of mitosis by glycopeptide dendrimer conjugates of colchicine

Glycopeptide dendrimers have been prepared bearing four or eight identical glycoside moieties at their surface (beta-glucose, alpha-galactose, alpha-N-acetyl-galactose, or lactose), natural amino acids within the branches (Ser, Thr, His, Asp, Glu, Leu, Val, Phe), 2,3-diaminopropionic acid as the branching unit, and a cysteine residue at the core. These dendrimers have been used as drug-delivery devices for colchicine. Colchicine was attached to the dendrimers at the cysteine thiol group through a disulfide or thioether linkage. The biological activities of the glycopeptide dendrimer conjugates were evaluated in HeLa tumor cells and non-transformed mouse embryonic fibroblasts (MEFs). The concentrations of glycopeptide dendrimer drug conjugates required to achieve inhibition of cell proliferation by interference with the tubulin system were found to be higher (IC50 > 1 microM) compared to the required colchicine concentration. On the other hand, the glycopeptide dendrimer conjugates inhibited the proliferation of HeLa cells 20-100 times more effectively than the proliferation of MEFs. In comparison, non-glycosylated dendrimers and colchicine itself showed a selectivity of 10-fold or less for HeLa cells.     

Taming C60 fullerene: tuning intramolecular photoinduced electron transfer process with subphthalocyanines

Two subphthalocyanine–C₆₀ conjugates have been prepared by means of the 1,3-dipolar cycloaddition reaction of (perfluoro) or hexa(pentylsulfonyl) electron deficient subphthalocyanines to C₆₀. Comprehensive assays regarding the electronic features – in the ground and excited state – of the resulting conjugates revealed energy and electron transfer processes upon photoexcitation. Most important is the unambiguous evidence – in terms of time-resolved spectroscopy – of an ultrafast oxidative electron transfer evolving from C₆₀ to the photoexcited subphthalocyanines. This is, to the best of our knowledge, the first case of an intramolecular oxidation of C₆₀ within electron donor–acceptor conjugates by means of only photoexcitation.     

Dual-Responsive Alginate Hydrogel Constructed by Sulfhdryl Dendrimer as an Intelligent System for Drug Delivery

Intelligent stimulus-triggered release and high drug-loading capacity are crucial requirements for drug delivery systems in cancer treatment. Based on the excessive intracellular GSH expression and pH conditions in tumor cells, a novel glutathione (GSH) and pH dual-responsive hydrogel was designed and synthesized by conjugates of glutamic acid-cysteine dendrimer with alginate (Glu-Cys-SA) through click reaction, and then cross-linked with polyethylene glycol (PEG) through hydrogen bonds to form a 3D-net structure. The hydrogel, self-assembled by the inner disulfide bonds of the dendrimer, is designed to respond to the GSH heterogeneity in tumors, with a remarkably high drug loading capacity. The Dox-loaded Glu-Cys-SA hydrogel showed controlled drug release behavior, significantly with a release rate of over 76% in response to GSH. The cytotoxicity investigation indicated that the prepared DOX-loaded hydrogel exhibited comparable anti-tumor activity against HepG-2 cells with positive control. These biocompatible hydrogels are expected to be well-designed GSH and pH dual-sensitive conjugates or polymers for efficient anticancer drug delivery.     

New Azido Coumarins as Potential Agents for Fluorescent Labeling and Their “Click” Chemistry Reactions for the Conjugation with closo-Dodecaborate Anion

Novel fluorescent 7-methoxy- and 7-(diethylamino)-coumarins modified with azido-group on the side chain have been synthesized. Their photophysical properties and single crystals structure characteristics have been studied. In order to demonstrate the possibilities of fluorescent labeling, obtained coumarins have been tested with closo-dodecaborate derivative bearing terminal alkynyl group. Cu^I catalyzed Huisgen 1,3-dipolar cycloaddition reaction has led to fluorescent conjugates formation. The absorption–emission spectra of the formed conjugates have been presented. The antiproliferative activity and uptake of compounds against several human cell lines were evaluated.